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The Impact of the Skeletal Muscle-specific TβRⅡ Knockout on the Denervated Muscles

Author: WangXu
Tutor: FanMing; ChenXiaoPing
School: PLA Military Academy of Medical Sciences
Course: Pathophysiology
Keywords: TβRII skeletal muscle denervation atrophy
CLC: R746.4
Type: PhD thesis
Year: 2011
Downloads: 49
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Abstract

Growth and differentiation of muscles are ordered process.Skeletal muscle atrophy happens in some pathological and physiological conditions, including denervation, disuse, malignant tumor and hunger. Even though researchers have kept studying the mechanisms of muscles’development and atrophy, a lot are still obscure, limiting the clinical treatment. TGFβs are the very important factors to affect the development and differentiation of muscles.TβRII is the key point for TGFβ1,TGFβ2,TGFβ3 to act. At present, how TGFβ-TβRII-SMAD3 affect the growth, differentiation and atrophy of muscles is not very clear.Here,the problem was studied using the TβRII knockout mice.TβRII flox/flox mice were generated using embryonic stem cell technology.The MCK-Cre mice were engineered containing Cre recombinase under control of ckmm muscle-specific promoter. TβRII flox/flox mice were crossed to MCK-Cre mice to generate TβRIIflox/wt/MCK-Cre double Tg mice. And then, TβRIIflox/wt/MCK-Cre double Tg mice were crossed with TβRII flox/flox mice to generate TβRIIflox/flox/MCK-Cre mice genetically ablating TβRII in cre-expressing skeletal muscle cells. As predicted, mice lacking TβRII by gene targeting in skeletal muscles were generated first in the world using Cre/loxP system. TβRII null mutant mice were viable, fertile and showed apparently normal development.We tested the TβRII expression level in different muscles by Real time PCR technology. The expression level of the TβRII in soleus,plantaris,gastrocnenmius lowered obviously. Statistical analysis was presented by comparing knockout mice and the control. The statistics showed that there was no significant difference between them.Depending on its fiber types, skeletal muscles can be divided into fast muscles which are represented by plantaris and slow muscles which represented by soleus.Because the single cross-section area of a fiber is an important index about atrophy of muscle, we totally separated the soleus and the plantaris of the knockout and the control mice. CSA of Muscle fibers were calculated by using Image-Proplus6.0 software and the result showed that the CSA of muscle fibers had no statistical difference between control mice and the knockout mice. Growth and differentiation of muscle can be adjusted by specific gene, so it encourages us to study how TβRII influence MRFs. Result showed that in the fast muscle, MRFs were increased obviously, including Myogenin(19.18times), MyoD(4.18 times), MRF4(4times), Myf(2.8times).In order to research the influence of TGFβ-TβRII-SMAD3 signaling pathway, we adopted the model of denervation.Frozen muscle tissues were selected for HE to observe the change of the CSA in single muscle fiber. It showed that after 14 days’denervation the CSAof the soleus fell 36.17% when compared with sham and control mice fell 10.64% (p < 0.01).The CSA of the knockout mice in plantaris fell 38.29% compared with sham while the control mice only fell 21.12% (p < 0.05).Under the condition of denervation ,the absence of TβRII promoted the atrophy.We then studied about their potential molecular mechanisms. Results showed that under the condition of denervation, the knockout of TβRII had little impact on the expression of Myogenin,MyoD,MRF4,Myf5. But compared with above we can speculate that the actions of TGF beta 1, TGF beta 2, TGF beta 3 factors are enfluenced by nerve distribution.AKT - P70S6K signaling pathway is the important way influencing the protein synthesis. The high expression of Atrogin -1 and MuRF-1 can cause muscle atrophy. The results showed that the the phosphorylation of P70S6K was significantly inhibited in soleus in the knockout mice. For the plantaris, the kocokout of TβRII obviously up-regulated the expression of Atrogin– 1. Both in the soleus muscle and plantaris, the knockout of TβRII had no obvious effect on the ratio of Bax to Bcl2.We went on studying the effects of the knockout of TβRII on the muscle fibers’conversion in the state of denevation. The results showed that the metabolism of both the quick twitch muscle and the slow twitch muscle had the trend to the direction of aerobic oxidation after knockout, meanwhile, the expression of the IIa,IIx muscle fibers contents in slow twitch muscle decreased, while the expression of the type I muscle fibers contents in quick twitch muscle increased 2.9 times.To further testify the study clue obtained from our experiments and to deepen the research better in the future , the promoter regions of genes of the key regulatory proteins having much to do with muscle atrophy and muscle fibers’conversion were blasted against the published genomic database and some SMAD3-binding regions were found out. To permit the TβRII to express again in the skeletal muscle, we constructed TβRII plasmids. In all, 15 protein-expressing plasmids and luciferase plasmids relevant to SMAD3 signal pathway were constructed.In conclusion, we developed the skeletal-specific T?RII Knockout mice first in the world ,constructed the denevated muscular atrophy model using the Knockout Mice, Observed the Impact of the TβRII knockout on the denervated atrophy .we found that the knockout of TβRII promoted the muscle fibers’atrophy in the state of denevation ,meanwhile influenced muscle fibers’conversion.

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